Investigation of Bacterial Community Composition and Abundance in a Lowland Arable Catchment
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Investigation of bacterial community composition and abundance in a lowland arable catchment A thesis submitted to the School of Environmental Sciences of the University of East Anglia in partial fulfilment of the degree of Doctor of Philosophy By Ali Khalaf A. Albaggar 2014 © This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with the author and that no quotation from the thesis, nor any information derived therefrom, may be published without the author’s prior consent. Abstract This study aimed to characterise the bacterial community composition and abundance in the River Wensum in Norfolk using epifluorescence microscopy (EFM), automated ribosomal intergenic analysis (ARISA) and 454 pyrosequencing. It also aimed to determine the effects of spatial and temporal variations and environmental factors on bacterial community composition and abundance in this intensively farmed lowland catchment. The three techniques provided the same trends in bacterial community composition and abundance across the Wensum catchment. Total bacterial numbers ranged from 0.21 × 10 6 cells/mL to 5.34 × 10 6 cells/mL (mean = 1.1 × 10 6 cells/mL). The bacterial community composition and abundance showed significant differences between sites and times and were related to environmental parameters, with temperature and flow rate explaining most of the variation in bacterial community composition and abundance. Bacterial abundance increases as water moves downstream, while bacterial diversity decreases as water moves downstream. Some operational taxonomic units (OTUs) become commoner as the water moves downstream (3 rd and 4 th order streams). This presumably reflects the fact that these bacteria are actively growing in the river, and reducing the abundance of other taxa. Consequently, the community becomes less diverse moving downstream, although a small number of sites do not fit this pattern. The River Wensum is dominated by the phyla Proteobacteria, Bacteroidetes, Cyanobacteria and Actinobacteria. Members of these phyla are well known to be responsible for biogeochemical processes, such as nitrogen cycling. The commonest bacteria at upstream sites were Proteobacteria (OTUs 2 and 4), Deltaproteobacteria (OTU29), Gammaproteobacteria (OTU32), Sphingobacteria (OUT9) and Flavobacteria (OTUs 12 and 23). Most OTUs (2, 9, 17, 29 and 32) are considered to be soil bacteria, suggesting that these bacteria are terrestrial in origin and are flushed into the lower order streams. Most of the upstream bacteria showed positive relationships with total nitrogen (TN) and total carbon (TC) and the presence of arable areas. On the other hand, the commonest bacteria at downstream sites were Cyanobacteria (OTU1), Flavobacteria (OTUs 3, 10 and 19), Cytophagia (OTU14), Actinobacteria (OTUs 20, 21 and 25) and Alphaproteobacteria (OTU26). Most of the downstream bacterial OTUs showed a positive relationship with TP and the presence of urban areas. The results of this research, however, do not provide strong evidence that competition is an important process structuring these bacterial communities. In addition, the correlations between environmental parameters and bacterial composition and abundance are not strong and do not clearly distinguish the most impacted sites from others. This suggests that bacterial community composition cannot be used as an indicator of the ecological status to assess compliance with Water Framework Directive (WFD) in a moderately impacted lowland catchment like the Wensum. ii Acknowledgments I would like to thank my supervisors Prof. Kevin Hiscock and Prof. Alastair Grant for giving me the opportunity of doing my PhD research under their respected supervision. Thank you for all your support, encouragement and advice. Without your support this work would not have been completed. I have no words to express my gratitude to you. Thank you very much to all UEA technicians including Mrs Judith Mayne, Mr Rob Utting and Miss Emily Sear for providing me with equipment and consumables support. Thank you very much to Mrs Jenny Stevenson, Mr Richard Cooper, Dr Christopher Adams and Dr Gilla Sünnenberg for all their help in the collection of samples. Many thanks also to Dr Tobias Krueger and Dr Faye Outram for providing me with environmental data relating to the Wensum catchment. I also want to thank Dr Amy Kirkham and Dr Jan Strauss for providing instrument support in the Oceanographic Marine Laboratory. I wish to thank the government of Saudi Arabia for providing me with the scholarship to undertake my project in the School of Environmental Sciences at the University of East Anglia. I am very grateful to all my family including my father, mother, sisters and brothers for all their support and encouragement. I would like to thank my wife, Soad Alghamdi, my daughters, Wejdan, Dalya and Dena and my son, Rayyan for their patience and support. My family has been a constant source of love, concern and strength all these years. Thank you to all my friends and colleagues at UEA including Mr Lewis Peake, Miss Amy Thomas, Mr Neill Mackay, Mr Robert Tickner and Miss Sian Foch-Gatrell. They have been a great source of encouragement and made my student years most enjoyable. iii List of Chapters Abstract ii Acknowledgements iii List of Tables x List of Figures xiii List of Abbreviations xvii Chapter One: General introduction 1 1.1 The quality of river water and the study of microbes 1 1.2 The study of bacteria in aquatic environments - why it is so important? 3 1.3 Bacterial diversity and structure 4 1.4 Total bacterial number and total heterotrophic bacteria 6 1.5 The effects of spatial and temporal variations (biogeography) on bacterial communities 10 1.5.1 Introduction 10 1.5.2 Spatial factors 11 1.5.2.1 Land use and bacterial communities 12 1.5.2.1.1 Plant-based agricultural practices and bacterial communities 13 1.5.2.1.2 Animal-based agricultural practices and bacterial communities 13 1.5.2.1.3 Urban areas and bacterial communities 14 1.5.3 Temporal factors 15 1.6 The effects of physical, chemical and biological characteristics on bacterial communities 17 1.6.1 Introduction 17 1. 6.2 Physical factors 18 1.6.2.1 Temperature 18 1.6.2.2 Rainfall 20 1.6.2.3 River flow 20 1.6.2.4 Total suspended solids (TSS) and free-living bacteria vs. particle-attached bacteria 20 iv 1.6.2.5 Residence time 21 1.6.3 Chemical factors 21 1.6.3.1 pH 21 1.6.3.2 Trophic nutrient status and bacterial communities 22 1.6.3.3 Bacterial tolerance to chemical pollutants 24 1.6.3.4 The role of bacterial communities in the bioremediation 24 1.6.4 Biological factors and bacterial communities 25 1.7 Thesis aims and objectives 26 1.8 Thesis outline 27 Chapter Two: A critical review of traditional and molecular techniques used for determining bacterial abundance and composition 28 2.1 Introduction to techniques for determining bacterial abundance 28 2.2 Culture methods and bacterial abundance 28 2.2.1 MPN technique 29 2.2.2 HPC technique 29 2.2.2.1 The media 30 2.2.2.1.1 Plate count agar (PCA) 31 2.2.2.1.2 R2A media 31 2.2.3 Disadvantages of culture-dependent methods 32 2.3 Microscopy 33 2.3.1 Epifluorescence microscopy (EFM) 34 2.3.2 Fluorochromes 35 2.3.2.1 DAPI (4', 6-diamidino-2-phenylindole) 35 2.3.2.2 DAPI concentration 36 2.3.2.3 Ideal time for staining with DAPI 37 2.3.2.4 Factors affecting the stability of DAPI 37 2.3.2.5 Acridine Orange (AO) 38 2.3.2.6 LIVE/DEAD BacLight TM kits. 38 2.3.2.7 Ethidium bromide (EB) 39 2.3.3 Scanning electron microscopy (SEM) 39 2.3.4 Flow cytometry (FCM) 40 v 2.3.5 Solid-phase cytometry 41 2.3.6 Fluorescent in situ hybridization (FISH) 41 2.3.7 Other uncommon enumerating methods 42 2.3.8 Filters and filtration processes 42 2.3.9 Fixation and preservation of water samples 43 2.3.10 Researcher bias 43 2.4 Molecular-based techniques for studying bacterial communities 45 2.4.1 Culture-dependent methods 45 2.4.2 DNA based characterisation of bacterial communities 45 2.4.2.1 DNA extraction methods 46 2.4.3 Non-PCR based methods 46 2.4.3.1 DNA reassociation 46 2.4.3.2 FISH 47 2.4.4 PCR-based methods 47 2.4.4.1 PCR technique 47 2.4.4.2 Genetic fingerprinting techniques 47 2.4.4.2.1 RISA/ARISA 48 2.4.4.2.2 DGGE and TGGE 51 2.4.4.2.3 SSCP (single strand confirmation polymorphisms) 51 2.4.4.2.4 ARDRA (Amplified ribosomal DNA restriction analysis/ restriction fragment length polymorphism (RFLP) 52 2.4.4.2.5 Terminal restriction fragment length polymorphism (T-RFLP) 52 2.4.4.2.6 Repetitive extragenic palindromic-PCR (Rep-PCR) 53 2.5 Bacterial communities identification using next generation methods 55 2.5.1 16S rRNA gene marker, cloning and sequencing 55 2.5.2 Metagenomic approaches 56 2.5.3 Next generation sequencing 57 2.6 Tools and methods used in this research 60 Chapter Three: Determining total bacterial numbers in the River Wensum using epifluorescence microscopy and heterotrophic plate counts 62 3.1 Introduction 62 vi 3.2 Aims 63 3.3 Materials and methods 63 3.3.1 Study sites 63 3.3.2 Total bacterial numbers 65 3.3.2.1 Sample collection for measuring total bacterial numbers 65 3.3.2.2 Sample collection for physiochemical measurements and other environmental data 65 3.3.2.3 DAPI staining and filtration 66 3.3.2.4 Quantifying total bacterial numbers 67 3.3.3 Total heterotrophic bacterial counts 67 3.3.3.1 Medium preparation 67 3.3.3.2 Sample collection and dilution 67 3.3.3.3 Spread plate method 68 3.3.4 Statistical analysis of bacterial abundance and total heterotrophic bacteria 68